1. Field of the Invention
This invention relates to the detection of molecular interactions between biological molecules. Specifically, the invention relates to methods for producing apparatus for detecting molecular interactions between probe molecules immobilized on a microarray and target molecules exposed to the microarray. The methods of the invention can be used to provide microarrays of oligonucleotides for performing nucleic acid hybridization assays to detect molecular interactions between the oligonucleotides on the microarray and nucleic acid target molecules obtained or produced from a biological sample. The invention also provides the apparatus produced using the inventive methods, and methods for performing assays using said apparatus for detecting molecular interactions between probe and target biomolecules.
2. Background of the Invention
Immobilization of DNA, RNA, peptides, and other biomolecules through chemical attachment to a solid support or within a matrix has become an important method of molecular biology and pharmacological research and clinical diagnostics. It is especially important in the manufacturing of microarray or chip-based technologies. Microarrays have numerous applications, including diagnosis of disease, drug discovery, and genetic screening, among others.
Microfabricated arrays (biochips) of oligonucleotides and nucleic acids have utility in a wide variety of applications, including DNA and RNA sequence analysis, diagnostics of genetic diseases, gene polymorphism studies, and analysis of gene expression. In the process of biochip fabrication, large numbers of probe molecules are bound to small, defined regions of a substrate. Biochip substrates can comprise a number of substances, including glass slides, silicon wafers, or polymeric hydrogels. The chemistries that may be employed to immobilize probe molecules to a biochip array are limited to those that will be compatible with a chosen substrate and further are limited to those chemistries that will permit efficient attachment of a large number of different probe molecules to a single supporting substrate surface.
Conventional methods for attaching a biomolecule to a surface involve multiple reaction steps, often requiring chemical modification of the solid support itself, or secondary substrates such as hydrogels attached to a solid support, in order to provide an appropriate chemical functionality capable forming a covalent bond with the biomolecule. The efficiency of the attachment chemistry and strength of the chemical bonds formed are critical to the fabrication and ultimate performance of the microarray.
For arrays comprising polyacrylamide or other hydrogels, the necessary attachment functionality is currently provided by chemical modification of the hydrogel itself: amide, ester, or disulfide bonds are formed between probe molecules and the polymeric constituents of the hydrogel after polymerization and crosslinking of the hydrogel. An unresolved problem with this approach is that the attachment chemistry is not optimally stable over time, especially during subsequent manufacturing steps, and under typical conditions of use, where the microarray is exposed to high temperatures, ionic solutions, and multiple wash steps. As a consequence, the number of probe molecules on the array can be depleted during use, thus reducing the performance and limiting the useful life of the array. In addition, these methods have an inherently low coupling efficiency.
An alternative method is to covalently link an amine-terminated oligonucleotide to an available aldehyde moiety in the hydrogel matrix. In this reaction scheme, the initial product of the amine-aldehyde reaction is a chemically-reversible Schiff base. In order to stablize the bond between the solid support and the oligonucleotide, however, the Schiff base must be reduced. This has conventionally been achieved using a borane-pyridine complex dissolved in chloroform. However, a major disadvantage of this method for producing a stable covalent bond between the amine nitrogen and the aldehyde carbon atom is that the reaction must be performed as a three-phase reaction (the solid support, the aqueous hydrogel, and the organic borohydride). The triphasic nature of this reaction considerably lowers the reaction rate and yield of the reduction.
There is thus a need in the art for novel biochip arrays using different means and methods for attaching and stabilizing probe molecules to substrates. In particular, there remains a need in the art for methods that more straightforwardly and efficiently provide a stable chemical bond between amine-terminal oligonucleotide and hydrogel polymers on biological microarrays.
This invention provides methods for stabilizing amine-derivatized oligonucleotides and nucleic acids to aldehyde-functionalized solid supports. In preferred embodiments, the solid support comprises a microarray as defined herein, and most preferably the microarray further comprises a hydrogel matrix. The invention provides improved methods for forming a stable amine bond between an amine-containing biomolecules, most preferably an amino-terminal oligonucleotide or nucleic acid, and an aldehyde moiety comprising the polymeric component of the hydrogel. In preferred embodiments, the invention provides improved methods involving two phases (solid and liquid) to reduce an unstable Schiff base formed by contacting a solution of the amino-terminal oligonucleotide or nucleic acid with a hydrogel comprising an aldehyde moiety. In preferred embodiments, sodium cyanoborohydride is used to reduce the Schiff base. Microarrays produced using the methods of the invention and methods of use thereof are also provided.
Sodium cyanoborohydride is advantageously used to reduce the unstable Schiff base because this reagent is soluble in aqueous solutions and it is more effective than reductants known in the prior art. The methods provided by the invention are dramatically increased in reaction rate and yield of the stable amine adduct. In addition, sodium cyanoborohydride can be dispensed simultaneously with DNA oligonucleotides because of its aqueous solubility, does not adversely affect oligonucleotide integrity, or interfere with reaction of the terminal amine with the bound aldehyde. These features eliminate the requirement for performing a separate reduction step, which is not only time consuming but also hazardous. Further, these features facilitate automation of microarray manufacturing.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.